Polynucleotides encoding human EPO-FC fusion proteins with prolonged half-life and enhanced erythropoietic activity in vivo

ABSTRACT

A recombinant fusion protein comprising a human erythropoietin peptide portion linked to an immunoglobulin peptide portion is described. The fusion protein has a prolonged half-life in vivo in comparison to naturally occurring or recombinant native human erythropoietin. In one embodiment of the invention, the protein has a half-life in vivo at least three fold higher than native human erythropoietin. The fusion protein also exhibits enhanced erythropoietic bioactivity in comparison to native human erythropoietin. In one embodiment, the fusion protein comprises the complete peptide sequence of a human erythropoietin (EPO) molecule and the peptide sequence of an Fc fragment of human immunoglobulin IgG1. The Fc fragment in the fusion protein includes the hinge region, CH2 and CH3 domains of human immunoglobulin IgG1. The EPO molecule may be linked directly to the Fc fragment to avoid extraneous peptide linkers and lessen the risk of an immunogenic response when administered in vivo. In one embodiment the hinge region is a human Fc fragment variant having a non-cysteine residue at amino acid 6. The invention also relates to nucleic acid and amino acid sequences encoding the fusion protein and transfected cell lines and methods for producing the fusion protein. The invention further includes pharmaceutical compositions comprising the fusion protein and methods of using the fusion protein and/or the pharmaceutical compositions, for example to stimulate erythropoiesis in subjects in need of therapy.

RELATED APPLICATION

This application is a divisional of application Ser. No. 11/340,661filed 27 Jan. 2006, now U.S. Pat. No. 7,625,564.

TECHNICAL FIELD

This application relates to human erythropoietin fusion proteins.

BACKGROUND

Human erythropoietin (EPO), a member of the haematopoietic growth factorfamily, is synthesized mainly in the adult kidney and fetal liver inresponse to tissue hypoxia due to decreased blood oxygen availability[1]. The principal function of EPO is to act directly on certain redblood cell (RBC) progenitors and precursors in the bone marrow tostimulate the synthesis of hemoglobin and mature RBCs. It also controlsthe proliferation, differentiation, and maturation of RBCs. RecombinantEPO having the amino-acid sequence of naturally occurring EPO has beenproduced and approved to treat anemia associated with kidney functionalfailure, cancer and other pathological conditions [2]. In addition toits erythropoietic properties, recent research reports [3] indicate thatEPO also acts on non-bone marrow cells such as neurons, suggesting otherpossible physiological/pathological functions of EPO in the centralnervous system (CNS) and other organs/systems. Since EPO receptors havebeen found in many different organs, EPO may have multiple biologicaleffects, such as acting as an anti-apoptotic agent.

Human EPO is a glycoprotein with a molecular weight of 30.4 kilodaltons.Carbohydrates account for approximately 39% of its total mass. The EPOgene is located on chromosome 7q11-22 and spans a 5.4 kb region withfive exons and four introns [4]. The precursor of EPO consists of 193amino acids. Cleavage of the leader sequence and the last amino acid Argby post-translational modification yields the mature EPO having 165amino acids. Glycosylation, with three N-linked sites at Asn 24, Asn38,Asn83 and one O-linked site at Ser126, plays a crucial role in thebiosynthesis, tertiary structure and the in vivo bioactivity of EPO [5].EPO functions by binding to an erythropoietin receptor, a glycosylatedand phosphorylated transmembrane polypeptide with the molecular weightof 72-78 kilodaltons. This binding triggers the homodimerization of thereceptors that leads to the activation of several signal transductionpathways: JAK2/STAT5 system, G-protein, calcium channel, and kinases.Two molecules of EPO protein are needed to bind simultaneously to onereceptor molecule to achieve optimal receptor activation [6].

As the first hematopoietic growth factor approved for human therapy,recombinant human EPO (rHuEPO) has been used for the treatment of anemiaresulting from chronic renal failure, cancers (primarilychemotherapy-associated anemia), autoimmune diseases, AIDS, surgery,bone marrow transplantation and myelodysplastic syndromes, etc.Interestingly, recent studies have also observed that rHuEPO hasnon-blood system functions and shows the potential of being used as aneuroprotective drug for cerebral ischemia, brain trauma, inflammatorydisease and neural degenerative disorders [7].

Currently, three kinds of rHuEPO or rHuEPO analogs are commerciallyavailable, namely rHuEPO alpha, rHuEPO beta, and darbepoetin alfa [8].These three recombinant proteins bind to the same erythropoietinreceptor, but differ in structure, degree of glycosylation,receptor-binding affinity and in vivo metabolism. Since the initialintroduction of rHuEPO-alpha in the 1980s, clinicians quickly recognizedthe frequent dose/injection requirement of the drug as a significantshortcoming. The mean in vivo half-lives of rHuEPO alpha and rHuEPO betaadministered intravenously or subcutaneously are only 8.5 and 17 hoursrespectively [9, 10]. Patients therefore need an injection schedule ofdaily, twice weekly or three times per week which imposes a burden onboth patients and health care providers. Thus, there has been alongstanding need to develop recombinant EPO analogs having a longer invivo half-life and/or enhanced erythropoietic activity.

Attempts have been made in the prior art to genetically change orchemically modify the structure of the native EPO protein to either slowdown its in vivo metabolism or improve its therapeutic properties. Forexample, there appears to be a direct correlation between the amounts ofsialic acid-containing carbohydrates on the EPO molecule and its in vivometabolism and functional activity. Increasing the carbohydrate contentof the EPO molecule thus results in a longer half-life and enhancedactivities in vivo [11, 12]. Amgen has designed the rHuEPO analogdarbepoetin alpha to include 5 N-linked carbohydrate chains, two morethan rHuEPO. Darbepoetin alpha is also known as Novel ErythropoiesisStimulating Protein (NESP) and is sold under the trademark Aranesp™.Darbepoetin alpha differs from native human EPO at five positions(Ala30Asn, His32Thr, Pro87Val, Trp88Asn, Pro90Thr) which allows for theattachment of two additional N-linked oligosaccharides at asparaginesresidue positions 30 and 88. Darbepoetin alpha binds to the EPO receptorin an identical manner as native EPO to induce intracellular signalinginvolving tyrosine phosphorylation by JAK-2 kinase and the sameintracellular molecules Ras/MAP-k, P13-k and STAT-5. Due to theincreased carbohydrate content, the half-life of darbepoetin alpha inboth animals and humans is almost three fold-longer than that ofrHuEPO-alpha (25.3 hours vs 8.5 hours) [9]. Darbepoetin alpha (Aranesp™)also appears to exhibit enhanced bioactivity in comparison to naturallyoccurring or recombinant human EPO in vivo [13] and has been approved byFDA as a second generation rHuEPO drug; this drug only needs to beadministrated once per week to achieve the identical therapeutic effectsof 2-3 time injections per week of rHuEPO[10, 14, 15].

Other attempts to extend the half-life of EPO have focused on increasingthe molecular weight of the EPO protein through chemical conjugationwith polyethylene glycol (PEGylation) and the like. PEGylated-EPO has amuch larger molecular weight and is protected from being cleared fromcirculation and therefore has a longer plasma half-life [16]. However,PEGylation may alter the protein structure resulting in unanticipatedchanges of function and specificity of the EPO moiety. There are alsoreports of increasing the molecular weight of EPO by other methods, suchas to link the EPO molecule to a carrier protein (human albumin), or toform the homodimerization of two complete EPO molecules by using linkingpeptides (3- to 17-amino acids) or by chemical cross-linking reagents[17, 18, 19, 20]. While all these methods have achieved some success inextending the half-life and enhancing the activities of EPO, combiningthe EPO molecule with the Fc fragment of human immunoglobulin (IgG) in afusion protein as described in the present application achieves uniqueadvantages.

Human immunoglobulin IgG is composed of four polypeptides linkedcovalently by disulfide bonds (two identical copies of light chain andheavy chain). The proteolysis of IgG molecule by papain generates twoFab fragments and one Fc fragment. The Fc fragment consists of twopolypeptides linked together by disulfide bonds. Each polypeptide, fromN- to C-terminal, is composed of a hinge region, a CH2 domain and a CH3domain. The Fc fragment structure is almost the same among all subtypesof human immunoglobulin. IgG is among one of the most abundant proteinsin the human blood and makes up 70 to 75% of the total immunoglobulinsin human serum. The half-life of IgG in circulation is the longest amongall five types of immunoglobulin and may reach 21 days.

Modern bio-engineering technology has been successfully applied to thecreation of fusion proteins consisting of therapeutic protein fragments,such as cytokines and soluble receptors, and the Fc fragment of humanIgG [21, 22, 23, 24]. These fusion proteins have a significantly longerin vivo half-life while retaining their biological and therapeuticproperties. So far two fusion proteins comprising an Fc fragment havebeen successfully developed as biomedicines and approved by FDA for thetreatment of rheumatoid arthritis and chronic plaque psoriasis [25, 26].

It has been shown in the prior art that dimers of two EPO moleculeslinked either by chemical cross-linking or by a polypeptide exhibitenhanced in vivo activities and a prolonged half-life[17, 19]. Theenhanced activity may due to the more efficient binding of the EPO dimerto one receptor, and the prolonged in vivo half-life due to the largersize of the dimer protein. However, the chemical cross-linking processis not efficient and is difficult to control. Moreover, the linkagepeptide in the dimer of EPO may alter the three-dimensional structure ofEPO molecule and the peptide itself may stimulate immunogenic responsesin vivo. These shortcomings impair the therapeutic potential of EPOdimers, particularly since EPO replacement therapy in renal patients islife-long.

The need has therefore arisen for EPO analogs that have a significantlylonger half-life and enhanced erythropoietic activities in vivo but haveno increased immunogenic properties.

SUMMARY OF THE INVENTION

In accordance with the invention, a recombinant fusion proteincomprising a human erythropoietin peptide portion linked to animmunoglobulin peptide portion is described. The fusion protein has aprolonged half-life in vivo in comparison to naturally occurring orrecombinant native human erythropoietin. In one embodiment of theinvention, the protein has a half-life in vivo at least three foldhigher than native human erythropoietin. The fusion protein may alsoexhibit enhanced erythropoietic bioactivity in comparison to nativehuman erythropoietin.

In one embodiment of the invention the immunoglobulin peptide portion isan Fc fragment, such as an IgG1 fragment. The Fc fragment includes CH2and CH3 domains and a hinge region. The EPO peptide portion may bedirectly linked to the hinge region. Preferably the hinge region is atleast 9 amino acids in length. In one embodiment, the EPO peptideportion has a cysteine residue proximate the C terminal thereof and thehinge region includes a cysteine residue located nearest the EPO peptideportion. Preferably these two cysteine residues are spaced at least 12amino acids apart. In one embodiment, the EPO peptide portion maycomprise a complete EPO molecule directly linked to the immunoglobulinportion (i.e. no external peptide linkers are interposed between the EPOand immunoglobulin portions).

The invention also relates to multimeric protein constructs comprisingmultiple units of the fusion protein of the invention. For example, twofusion proteins may be assembled as a dimer, wherein the hinge regionsof the proteins are joined by disulphide bonds. The dimer has thegeneral shape of a IgG molecule and is more stable than free EPOmolecules.

The invention also relates to nucleic acid and amino acid sequencesencoding the fusion protein and transfected cell lines and methods forproducing the fusion protein. The invention further includespharmaceutical compositions comprising the fusion protein and methods ofusing the fusion protein and/or the pharmaceutical compositions, forexample to stimulate erythropoiesis in subjects in need of therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

In drawings which illustrate various embodiments of the invention butwhich are not intended to be construed in a limiting manner:

FIG. 1A is a schematic diagram showing the general structure of therecombinant human EPO-Fc fusion protein (rHuEPO-Fc) of the invention.

FIG. 1B is a sequence listing showing the nucleotide sequence and thededuced amino-acid (aa) sequence of rHuEPO-Fc protein. The total lengthof DNA is 1281 bp. The 426 amino acids in the deduced protein sequenceinclude 27 aa for the signal peptide and 399 aa for the completerHuEPO-Fc protein. The complete rHuEPO-Fc protein consists of human EPOdomain (166aa), hinge region (16aa, underlined), and CH2 and CH3 domains(217 aa) of the Fc fragment of human IgG1. The calculated molecularweight of the polypeptide of the mature rHuEPO-Fc fusion protein is 44.6kDa, composed of 18.5 kDa (41.4%) of EPO fragment and 26.1 kDa (58.6%)of IgG1 Fc fragment. A homodimer is formed by two disulfide bonds viathe two cysteine residues (boxed) within the hinge region. At residue172 of the mature fusion protein (i.e. the 6^(th) amino acid of hingeregion) the native cysteine residue has been substituted by glycine(bold).

FIG. 2 is a schematic diagram showing the structure and features of themammalian expression plasmid pCD1 used for inserting the DNA sequenceencoding the polypeptide of the rHuEPO-Fc fusion protein, and fortransfecting CHO cells that express the rHuEPO-Fc fusion protein.

FIG. 3 is a SDS-PAGE image showing the sizes of the dimeric form of purerHuEPO-Fc protein in non-reduced condition and monomeric form of purerHuEPO-Fc protein in reduced condition by SDS-PAGE analysis. Thepurified rHuEPO-Fc protein from the supernatants of the cultured CHOcell-line expressing rHuEPO-FC exists mainly as the dimeric form and hasa molecular weight of about 180 kDa on 8% Bis-Tris gel in non-reducedcondition (column A). In reduced condition (100 mM dithiothreitol, DTT)to break disulfide bonds, the dimer is separated into two identicalmonomeric units with a molecular weight of 75 kDa (column B).

FIGS. 4A and 4B are graphs showing the dose-dependent increase ofhemoglobin (Hb) levels in normal mice treated with three times per weeksubcutaneous injection (s.c.) of rHuEPO-Fc or rHuEPO. Each pointrepresents the mean Hb level of the group (6 mice). Day 0 levelsrepresent the Hb levels before treatment. A: Mice treated withrHuEPO-Fc. B: Mice treated with native rHuEPO

FIGS. 5A and 5B are graphs showing the dose-dependent increase ofhemoglobin (Hb) levels in normal mice treated with once per week s.c. ofrHuEPO-Fc or rHuEPO. Each point represents the mean Hb level of thegroup (6 mice). Day 0 levels represent the Hb levels before treatment.A: Mice treated with rHuEPO-Fc. B: Mice treated with native rHuEPO

FIGS. 6A and 6B are graphs showing the increase of hemoglobin (Hb)levels in normal mice treated with intravenous injection (i.v.) of 12.5μg/kg of rHuEPO-Fc or rHuEPO. Each point represents the mean Hb level ofthe group (6 mice). Day 0 levels represent the Hb levels beforetreatment. A: Mice with treatment once a week. B: Mice with treatment 3times a week.

FIG. 7 is a graph showing the dose-dependent increase of hemoglobin (Hb)levels in ⅚ nephrectomized rats treated with once per week s.c. ofrHuEPO-Fc, rHuEPO or darbepoetin-alfa (abbreviated Darbe.). Each pointrepresents the mean Hb level of the group. Normal controls were normalrats with injection of carrier solution. Model controls were the ⅚nephrectomized rats with injection of carrier solution. Week 0 levelsrepresent the Hb levels before treatment. *: week(s) post treatment.

FIG. 8 is a graph showing the dose-dependent increase of hemoglobin (Hb)levels in ⅚ nephrectomized rats treated once every two weeks s.c. withrHuEPO-Fc, rHuEPO or darbepoetin-alfa (abbreviated Darbe.). Each pointrepresents the mean Hb level of the group. Normal controls were normalrats with injection of carrier solution. Model controls were the ⅚nephrectomized rats with injection of carrier solution. Week 0 levelsrepresent the Hb levels before treatment. *: week(s) post treatment.

FIG. 9 is a graph showing the dose-dependent increase of hemoglobin (Hb)levels in ⅚ nephrectomized rats treated once every two weeks i.v. with62.5 μg/kg of rHuEPO-Fc, or darbepoetin-alfa (abbreviated Darbe.). Eachpoint represents the mean Hb level of the group. Normal controls werenormal rats with injection of carrier solution. Model controls were the⅚ nephrectomized rats with injection of carrier solution. Week 0 levelsrepresent the Hb levels before treatment. *: week(s) post treatment.

FIG. 10A-10C show the potency comparisons of rHuEPO-Fc, rHuEPO anddarbepoetin-alfa for stimulating the colony formation of CFU-E and BFU-Ein ⅚ nephrectomized rats treated with different doses and schedules.rHuEPO-Fc and darbepoietin-alpha (abbreviated Darbe.) treatment showedsimilar dose-dependent potencies for stimulating the CFU-E and BFU-Ecolony formation, while rHuEPO was less potent. A, s.c. once every week.B, s.c. once every 2 weeks. C., i.v. once every two weeks.

FIG. 11 is a graph showing the serum levels of rHuEPO-Fc and rHuEPOafter the intravenous injection of 5 μg/kg of rHuEPO-Fc or rHuEPO toRhesus monkeys (mean levels of 5 monkeys).

DETAILED DESCRIPTION OF THE INVENTION

Throughout the following description specific details are set forth inorder to provide a more thorough understanding of the invention.However, the invention may be practiced without these particulars. Inother instances, well known elements have not been shown or described indetail to avoid unnecessarily obscuring the present invention.Accordingly, the specification and drawings are to be regarded in anillustrative, rather than a restrictive sense.

This application relates to a novel fusion protein having erythropoieticproperties. The fusion protein, referred to herein as rHuEPO-Fc,comprises a human erythropoietin (EPO) molecule recombinantly linked toan immunoglobulin Fc fragment. As discussed further below, the fusionprotein may be in the form of a dimer consisting of two identicalpolypeptide subunits. In the embodiment shown schematically in FIG. 1A,each polypeptide subunit, from the N-terminal to C-terminal, consists ofthe polypeptide sequence of the human EPO molecule and the polypeptidesequence of the hinge region, CH2 domain and CH3 domain of the Fcfragment of human immunoglobulin IgG1. The two polypeptide subunits areconnected together by disulfide bonds between the respective hingeregions to form the dimer structure. The dimer thus has the same generalshape as an IgG molecule and exhibits better stability than free EPOmolecules as discussed in the examples below.

As will be apparent to a person skilled in the art, the hinge region ofan intact immunoglobulin provides the protein sufficient flexibility foreffective antigen-antibody binding. Similarly, in the present inventionthe hinge region is included in the design of the rHuEPO-Fc fusionprotein to maintain its flexibility, especially when the fusion proteinis in the dimer form. As described below, this allows the normal bindingof the EPO portion of the rHuEPO-Fc fusion protein to EPO receptors toactivate EPO biological functions. It is believed that the dimer form ofthe rHuEPO-FC fusion protein, by providing two EPO molecules, is capableof inducing the optimal activation of EPO receptors (for example, byfacilitating receptor cross-linking).

As demonstrated in the examples set forth below, the rHuEPO-Fc fusionprotein has been successfully synthesized using recombinant DNAtechniques. The fusion protein has been shown in mice, rat and primatestudies to exhibit a prolonged in vivo half-life and enhancederythropoietic properties in comparison to naturally occurring orrecombinant native human EPO. As used in this patent application, theterms “native human erythropoietin” and “native human EPO” mean EPOhaving an unmodified wild type structure. As will be appreciated by aperson skilled in the art, native human EPO may be naturally occurringor recombinantly produced (e.g. rHuEPO alpha). The term “native humanEPO” does not include rHuEPO analogs, such as darbepoetin alpha wherethe EPO structure has been significantly modified, such as byhyperglycosylation.

The nucleic acid sequence of the rHuEPO-Fc fusion protein of the presentinvention is shown in SEQ. ID. No. 1. The corresponding deduced aminoacid sequence is shown in SEQ. ID. No. 2. The complete rHuEPO-Fc fusionprotein is 399 amino acids in length. As shown in FIG. 1B, the completerHuEPO-Fc fusion protein consists of the EPO domain (166 amino acids),the hinge region (16 amino acids, underlined) and the CH2 and CH3domains (217 amino acids). A signal or leader peptide sequenceconsisting of 27 amino acids is also shown in FIG. 1B. The signalpeptide is cleaved during synthesis of rHuEPO-Fc. The nucleic and aminoacid sequences of rHuEPO-Fc including the signal or leader peptide areshown in SEQ. ID. No. 3 and SEQ. ID. No. 4 respectively.

As shown best in FIG. 1B and SEQ. ID. No. 2, the EPO domain has acysteine residue near the C-terminal thereof at amino acid number 161.The hinge region includes 2 cysteine residues, at amino acid numbers 178and 181 which are boxed in FIG. 1B. The hinge region cysteine residuesform the disulphide bonds between the polypeptide subunits of thehomodimer as discussed above. The naturally occurring hinge region of ahuman IgG1 fragment also has a cysteine at residue number 6 of the hingeregion portion (measured from the N-terminal). In the present invention,the cysteine residue 6 of the hinge portion has been substituted by anon-cysteine residue. In particular, in the embodiment of FIG. 1B andSEQ. ID. No. 2, the amino acid cysteine has been substituted by glycine(at amino acid residue 172 of rHuEPO-Fc, which corresponds to residue 6of the hinge region). As will be apparent to a person skilled in theart, other non-cysteine residues could also be substituted for cysteineat this location to avoid formation of a disulphide bond.

As a result of the amino acid substitution at residue 172, the firstcysteine residue of the hinge region (at residue 178) is spaced 17 aminoacids from the above-described cysteine residue of the EPO domain (atresidue 161). The inventors believe that the minimum spacing between thecysteine residue 161 of the EPO domain and the first cysteine residue ofthe hinge region should be at least 12 amino acids to enable successfulassembly and/or EPO receptor binding of a homodimer of rHuEPO-Fc. Thatis, if residue 172 is a cysteine residue, an undesirable disulphide bondmay potentially be formed, such as between cysteine residues 161 and172. This may alter the three dimensional structure of the EPO molecule,resulting in biological inactivity.

In one embodiment of the invention, the EPO domain is linked directly tothe Fc fragment portion of the fusion protein. By avoiding providing anexternal linker peptide, the preferred three dimensional structure ofthe rHuEPO-Fc fusion peptide is maintained and the risk of triggering anundesirable immunogenic response is minimized. The hinge region of theFc fragment is preferably at least 9 amino acids in length and ispreferably in the range of about 10-20 amino acids in length.

EXAMPLES

The following examples will further illustrate the invention in greaterdetail although it will be appreciated that the invention is not limitedto the specific examples.

1. Construction of the Recombinant Plasmid pCdEpo-Fc Encoding the FusionProtein of HuEPO-Fc.

The full length DNA molecule, which encodes the amino-acid sequence ofthe polypeptide of rHuEPO-Fc, was generated by overlapping PCR using thefollowing oligo primers (QIAGEN Inc., US):

EF5: 5′-ccggaattcgccaccatgggggtgcacgaatgtcctgcct-3′; EF3:5′-ttttccttttgcggccgcttatttacccggagacagggagag-3′; EFL5:5′-aggcctgcaggacaggggacagagttgagcccaaatctggtgac a-3′; EFL3:5′-tgtcaccagatttgggctcaactctgtcccctgtcctgcaggcc t-3′.The sequences of the above-noted primers are listed in SEQ. I.D. Nos.5-8 respectively.

EcoR I and Not I sites were introduced in EF5 and EF3, respectively. Foroptimal expression of the HuEPO-Fc protein in mammalian cells, the Kozaksequence (GCCACCATGG) was also introduced in EF5. EFL5 and EFL3 arecomplementary sequences consisting of 3′-terminal DNA sequence of Epo(23 bp) and 5′-terminal DNA sequence of IgG1 hinge (22 bp).

First, an EPO DNA fragment of 0.6 kb was amplified by PCR (Platinum TaqDNA Polymerase High Fidelity) with primers EF5 and EFL3 from plasmid p9Econtaining the full length of human EPO cDNA, Fc fragment of 0.7 kb withprimers EF3 and EFL5 from plasmid pD containing the full length of humanIgG1 cDNA sequence, respectively (p9E and pD are from the inventors' ownlab). The two fragments were then purified and mixed in equal amount.Using the mix as template, the full length rHuEPO-Fc DNA of 1.3 kb wasamplified by primers EF5 and EF3. The purified 1.3 kb fragment wasdigested by EocR I and Not I (New England Biolab Inc. US) and thencloned into EcoR I/Not I-digested mammalian expression vector pCD1 (FIG.2). The resulting recombinant vector was named pCdEpo-Fc and theinserted nucleic-acid sequence encoding the amino-acid sequence of theHuEPO-Fc protein was confirmed by DNA sequencing.

2. Establishment of rHuEPO-Fc Expression Cell Line

Chinese hamster ovary cell with dihydrofolate reductase (dhfr)deficiency (CHO/dhfr⁻, ATCC No. CRL-9096), which has been approved byFDA for biological substance production, was used as the host cell forrHuEPO-Fc expression.

The CHO-dhfr⁻ cells were transfected with the recombinant vectorpCdEpo-Fc using Lipofectamine (Gibco, Cat. No: 18292-037, USA). Thesupernatants from the culture of selected clones were assayed by ELISA(Roche, Cat. No: 1-693 417, Canada) for EPO activity. Positive cloneswere further screened under increasing Methotrexate (MTX) pressures. Onecell line with highest rHuEPO-Fc protein expression was selected as therHuEPO-Fc-expressing CHO cell-line, and gradually adapted to serum-freemedia (CD CHO Medium, Gibco, Cat. No: 10743-029, USA). ThisrHuEPO-Fc-expressing CHO cell-line was used for the production ofrHuEPO-Fc protein.

3. Purification of rHuEPO-Fc Protein

rHuEPO-Fc protein molecules contained in the supernatants collected fromthe serum-free media culturing the rHuEPO-Fc-expressing CHO cells wereisolated at first by Protein A affinity chromatography (Amersham, Cat.No: 17-0402-01, Canada). The isolated proteins were further purified bygel filtration in HiLoad 16/60 Superdex 200 pg column (Amersham, Cat.No: 17-1069-01, Canada). The purity of the rHuEPO-Fc protein was morethan 98% as determined by electrophoresis.

4. Determination of the Sizes of the Pure rHuEPO-Fc Protein

First, SDS-PAGE was carried out to determine the sizes of the purerHuEPO-Fc protein. As shown in FIG. 3, a single band with molecularweight of about 180 kDa was seen on 8% Bis-Tris gel in the non-reducedcondition, which measured the overall size of the protein with theexistence of disulfide bonds. This indicated that most rHuEPO-Fc proteinmolecules were produced as the dimeric form, as expected from the designof the fusion protein. When SDS-PAGE analysis was conducted in thereducing condition (100 mM dithiothreitol, DTT) to break the disulfidebonds, only the band with molecular weight of 75 Kda was identified,consistent with the estimated molecular weight of single polypeptidechain of HuEPO-hinge region-CH2-CH3.

The accurate molecular weight of the pure rHuEPO-Fc fusion protein withglycosylation, determine by Mass Spectrum (MALDI-TOF-MS), was 111099dolton (111.1 Kda). In this assay, only a single peak of protein wasobserved, indicating the purified rHuEPO-Fc protein was nearly 100%pure. The 15 amino acids of the N-terminal of the pure rHuEPO-Fc proteinwas determined by protein sequence analysis as: APPRLICDSRVLERY. Thiswas consistent with the sequence of the first 15 amino acids of thenative human EPO polypeptide, and confirms that the purified rHuEPO-Fcprotein does have the right and complete EPO molecule sequence aspredicted by the DNA sequence encoding the amino-acid sequences of therHuEPo-Fc fusion protein.

5. Enhanced Erythropoietic Activities of rHuEPO-Fc in Normal Mice

In vivo experiments in mice were conducted to confirm the retaining ofthe erythropoietic activity of the rHuEPO-Fc protein and determine itsefficacy compared to rHuEPO and darbepoetin-alpha. For comparisonpurpose, all the doses of three EPOs used in the described animalexperiments of the invention: our rHuEPO-Fc, rHuEPO (i.e. native humanEPO) and darbepoetin-alpha, were the amounts of EPO molecule portionalone based on the molar basis. In respect to rHuEPO-Fc protein, the EPOportion contributes to 41.4% of the total rHuEPO-Fc molecular weight ascalculated by the ratio of the weight of amino acids of EPO in theweight of the total amino acids of the whole rHuEPO-Fc molecule (166 aaamong 399 aa). The EPO amount for rHuEPO-Fc was then decided as 41.4% ofthe total amount of the rHuEPO-Fc protein.

rHuEPO-Fc (stock concentration: 0.5 mg/ml, purity of 98.6%) and nativehuman rHuEPO (i.e. with natural human EPO structure) (6000 IU/0.5 ml,manufactured by Kirin Brewery Co., Japan) were diluted in carriersolution (2.5 mg/ml of human serum albumin, 5.8 mg/ml of sodium citrate,0.06 mg/ml of citric acid and 5.8 mg/ml of sodium chloride, pH5.5-5.6).The dose of rHuEPO in amount was calculated according to itsactivity/amount ration. BALB/c mice (6- to 8-week old, weighing 18-22 g,equal numbers of male and female, purchased from Experiment AnimalCenter, AMMS, China) were grouped randomly with 6 in each group. Eachgroup of mice was treated with one combination of one dose (0.1, 0.5,2.5, 12.5, 62.5 μg/kg), one injection route (i.v. through the tail veinor s. c.) and one injection schedule (three times per week or once perweek). The control group of mice was injected with the equal volume ofcarrier solution. The treatment lasted for 3 weeks and the totalobservation times were 5 weeks. Peripheral blood samples (tail vein) formeasurement were taken before treatment, on the 4^(th) day and 7^(th)day of every week for 5 weeks. Hb was measured as the index byabsorptiometry. Mean±SD was calculated from the data of each group and ttest was conducted among different groups.

The administration of EPO three times per week to mice, provided thatthe EPOs have normal erythropoietic activity, would induce saturatedstimulation of erythropoiesis. As shown in FIG. 4, both groups treatedwith 3 times per week s.c. had significant elevation of Hb levels evenat the dose of 2.5 μg/kg. This experiment demonstrated that rHuEPO-Fcexhibited an in vivo erythropoietic activity as effective as rHuEPO. Theelevation of Hb levels in the treated group was dose-dependent. However,saturated elevation of the Hb levels was induced in mice at the dose of12.5 μg/kg of rHuEPO-Fc, whereas the similar saturated elevation of theHb levels was only achieved at the dose of 62.5 μg/kg of rHuEPO. Theelevation of Hb levels induced by 2.5 μg/kg of rHuEPO-Fc was alsogreater than that by 2.5 μg/kg of rHuEPO. These results suggested morepotent erythropoietic stimulation by rHuEPO-Fc than rHuEPO.

The erythropoietic potency of rHuEPO-Fc was further explored by reducingthe injection times to once per week subcutaneously. As shown in FIG. 5,the rHuEPO-Fc-treated groups showed dose-dependent elevation of Hblevels at the doses of 12.5, or 62.5 μg/kg. Both doses of 12.5 and 62.5μg/kg of rHuEPO also induced the elevation of Hb levels to the similarextent, which was much lower than that by 62.5 μg/kg of rHuEPO-Fc. Thisstrongly indicates that rHuEPO-Fc has enhanced erythropoietic activityin vivo. It is presumably due to either the prolonged half-life of therHuEPO-Fc in vivo or improved EPO receptor binding/activation by thedimer EPO molecules in the rHuEPO-Fc protein, or by the combined effectsof both.

When the same doses (12.5 μg/kg) of rHuEPO-Fc or rHuEPO wereadministrated intravenously either three times per week or once perweek, elevation of the Hb levels was observed for all the treated groups(FIG. 6). However, i.v. administration once per week of rHuEPO-Fcinduced greater, more persistent elevation of the Hb levels, whichcontinued longer after the treatment was over. This data providesfurther support for the enhanced erythropoietic properties of therHuEPO-Fc protein in comparison with rHuEPO having the structure ofnaturally occurring EPO protein.

6. Enhanced Erythropoietic Activities of rHuEPO-Fc in ⅚ NephrectomizedRats

Experiments in normal mice proved the enhanced erythropoietic activitiesof rHuEPO-Fc in vivo. To further observe the efficacy of rHuEPO-Fc instimulating erythropoiesis, pharmacodynamic studies were conducted inrats with experimental renal anemia that was made by ⅚ nephrectomy. Theefficacy of rHuEPO-Fc was compared with those of rHuEPO anddarbepoetin-alpha (60 μg/ml, lot. No. N079, manufactured by KirinBrewery Co., Japan).

Wistar rats (male and female in equal number, weighing 160-180 g,purchased from Vitalriver Experiment Animal Inc., Beijing, China.License No. SCXK11-00-0008) were used in this invention to create theanemia model due to the renal functional failure by a two-stepnephrectomy [27]. ⅚ nephrectomy was done to rats with general anesthesiaby two separate operations under sterile condition. After ⅔ of the leftkidney was resected, the rats were allowed to recover for twenty days.The right kidney was then resected carefully. Antibiotics wereadministrated to prevent infection after each operation. In total ⅚ ofthe kidney tissue was finally resected. The nephrectomized ratsgradually developed renal function dissufficiency and anemia. The ratsentered stable status of anemia 50 days after nephrectomy, and were thenrandomly grouped (9/group) to start the administration of the EPOs. Eachgroup of rats was treated with one combination of one dose (2.5, 12.5,62.5 μg/kg), one injection route (i.v. through the tail vein or s. c.)and one injection schedule (once per week or once every 2 weeks). Thecontrol group and model group of rats were injected with the equalvolume of carrier solution. The treatment lasted for 4 weeks and thetotal observation times were 6 weeks.

All doses (2.5, 12.5, 62.5 μg/kg) of rHuEPO-Fc, administeredsubcutaneously once per week, induced dose-dependent elevation of the Hblevels comparing to the model control group that did not receive EPOtreatment. Both 12.5 and 62.5 μg/kg of rHuEPO or darbepoetin,administrated subcutaneously once per week also induced elevation of Hblevels. The increased levels of Hb in both groups treated with 12.5 or62.5 μg/kg of rHuEPO-Fc were significantly higher than those in groupstreated with 12.5 or 62.5 μg/kg of rHuEPO respectively. The Hb levels in62.5 μg/kg of rHuEPO-Fc-treated groups were also slightly higher thanthat in 62.5 μg/kg of darbepoetin-treated group. After stoppingtreatment, the decrease of Hb levels in 62.5 μg/kg of rHuEPO-Fc-treatedgroup was much slower and the Hb levels remained higher than those ofboth normal control and model control groups until the end ofobservation (two weeks after treatment), indicating a stronger and/or aprolonged erythropoietic stimulation (summarized in FIG. 7).

For the treatment of subcutaneous injection once every two weeks, only12.5 or 62.5 μg/kg of the three EPOs were administered (FIG. 8). 12.5μg/kg of rHuEPO barely increased Hb levels compared to the model controlgroup, and the weak erythropoietic response in the 62.5 μg/kg of rHuEPOtreated group failed to bring the Hb levels to normal in comparison withthe normal control group. Treatments of either rHuEPO-Fc or darbepoetinat the doses of 12.5 or 62.5 μg/kg induced significant elevation of Hblevels that was higher than that of the normal control group, indicatingthe effective correction of anemia status by both rHuEPO-Fc anddarbepoetin. No significant differences were observed between same dosesof rHuEPO-Fc and darbepoetin in terms of efficacy. The high dose of 62.5μg/kg resulted in the persistent increase of erythropoiesis until thetermination of the observation (two weeks post treatment). This furthersuggested that rHuEPO-Fc and darbepoetin exhibit the property oflong-lasting stimulation of erythropoiesis in vivo, which in turn couldbe transferred to the reduction of administration frequencies topatients clinically.

While darbepoetin has been approved for clinical application withless-frequent injections to increase the patient compliance and reducethe work burden of health care providers, these experimental datastrongly indicate that rHuEPO-Fc disclosed in the current invention hasat least the similar potential benefits. As discussed above,darbepoetin, as a mutant analog of the human EPO molecule containingadditional sugar compounds (increased glycosylation), may have anincreased risk of inducing immunogenesis in vivo due to the alteredthree dimensional structures. Only long-term observation of patientsundergoing treatment with darbepoetin will give a decisive answer to theimmunogenic risks of darbepoetin. In contrast, rHuEPO-Fc, without themodification of the EPO molecule portion, has a carbohydrate contentidentical or closely similar to that of native human EPO. The amounts ofsialic acids in the inventors' pure rHuEPO-Fc protein were around 10.0mmol/mmol EPO, consistent with the reported parameters of rHuEPO. The Fcpart of rHuEPO-Fc, with no external amino acid(s)/linking peptide,represents the general structure of human IgG1, and theoretically wouldnot lead to an immunogenic response. If approved clinically, rHuEPO-Fcmay provide a better choice for patients than currently available rHuEPOand EPO analogs, especially those who need long-term administration.

Once injected intravenously once every two weeks, both rHuEPO-Fc anddarbepoetin (62.5 μg/kg) were able to induce identical increases of Hblevels in the rats with renal anemia far above the normal Hb levels inthe normal control rats (FIG. 9). This further demonstrates thepersistent stimulation of erythropoiesis by rHuEPO-Fc, as darbepoetin'sefficacy has been clinically proven.

Data derived from cell culturing experiments of bone marrow cellscollected from the ⅚ nephrectomized rats after treatments (once per weekor per two weeks, s.c. or i.v.) showed that rHuEPO-Fc, rHuEPO anddarbepoetin all stimulated the formation of CFU-E and BFU-E. Thepotencies of rHuEPO-Fc and darbepoetin were similar and stronger thanthat of rHuEPO (FIG. 10).

Blood urinonitrogen (BUN) and Crea levels were similar in the treatedgroups and model control group. The levels of serum Fe in all thetreated groups were higher that that of the model control group.Pathological examinations observed the increase distribution of redblood cell (RBC)-related cells in bone marrow and spleen of allEPO-treated rats.

7. Pharmacokinetic Studies of rHuEPO-Fc in Rhesus Monkeys

As discussed above, the inventors have designed rHuEPO-Fc in such waythat the EPO portion of the fusion protein retains the functionalproperties of natural EPO, such as stimulating erythropoiesis, and theFc fragment of human IgG1 allows the stable existence of the fusionprotein in circulation, thus extending its half-life in vivo. The aboveanimal studies have demonstrated the erythropoietic activities ofrHuEPO-Fc are enhanced in comparison with rHuEPO. The inventors havealso conducted pharmacokinetic studies to determine the in vivohalf-life of rHuEPO-Fc in comparison to that of rHuEPO. Primates wereused to generate data as they are biologically very similar to humanbeings.

Study design was based on literature reports and the experiments wereconducted according to the general guidelines of pharmacokinetics. Twogroups of Rhesus monkeys with 5 monkeys in each group (3-5 kg, purchasedfrom the Experiment Animal Center, AMMS, China) were injectedintravenously with 5 μg/kg of rHuEPO-Fc or rHuEPO, respectively. Bloodsamples were taken before and at 0.017, 0.167, 0.5, 1, 2, 4, 8, 12, 24,48, 96, 168, 240 h after injection. Sera were collected bycentrifugation and the serum rHuEPO-Fc or rHuEPO levels were determinedby using human erythropoietin enzyme-linked immunosorbent assay (ELISA)kits (purchased from R&D Systems, Minneapolis, Minn.). The averagehalf-life (t½) of rHuEPO-Fc and rHuEPO injected intravenously was35.24+/−5.15 h and 8.72+/−1.69 h respectively (summarized in FIG. 11).

To observe the bioavailability of rHuEPO-Fc, 5 μg/kg of rHuEPO-Fc wasinjected subcutaneously to 5 Rhesus monkeys. Blood samples were takenbefore and 1, 2, 5, 8, 10, 12, 15, 24, 48, 72, 96, 168, 240 h after theinjection, and the serum levels of rHuEPO-Fc were determined by the R&Dkits. The bioavailability index was calculated as 35.71+/−5.37% with thesubcutaneous injection. This is identical to the reportedbioavailability figures of darbepoetin-alpha (Aranesp™) in patients withchronic renal failure [9, 15].

This data demonstrates that rHuEPO-Fc has a significantly prolongedhalf-life in primates, and the in vivo half-life of rHuEPO-Fc is atleast four fold longer than that of rHuEPO manufactured by Kirin BeerBrewing Co. of Japan. The prolonged half-life in vivo likely contributesto the enhanced erythropoietic activity of rHuEPO-Fc.

8. Immunogenicity of rHuEPO-Fc in Macaca fascicularis

As indicated above, attention was given in the design of rHuEPO-Fcfusion protein to intentionally avoid or minimize the changes of theimmunogenic properties of the rHuEPO-Fc fusion protein. The inventorsavoided including/adding any external amino acid(s) or linking peptidesequences in the fusion protein. The invented HuEPO-Fc fusion protein ofthe embodiment of FIG. 1B only contains the polypeptide sequences of thenatural EPO protein and the Fc fragment (hinge region, CH2, CH3) ofhuman IgG1, and would theoretically not induce an immunogenic responseand the production of antibodies against rHuEPO-Fc protein. As will beappreciated by a person skilled in the art, other embodiments havingalternative structures are also encompassed by the present invention.

The following primate studies were conducted to observe theimmunogenicity of rHuEPO-Fc protein. Ten crab-eating macaque (Macacafascicularis) (male/female=5/5, ˜5 years old, average weight of male4.0±0.3 kg, female is 2.9±0.4 kg, purchased from Laboratory AnimalCenter, AMMS, China) were injected subcutaneously with 5 μg/kg ofpurified rHuEPO-Fc 3 times per week for 4 weeks, and two were injectedwith equal volume of carrier solution as the control animals. Sera werecollected once a week for 5 weeks (1 week post-treatment) and tested forthe specific antibodies against rHuEPO-Fc by ELISA using the purifiedrHuEPO-Fc (5 μg/ml) as the coating antigen. In addition, RBC count andHb levels in the peripheral blood were also determined within theexperimental period. The resultant data shows that, while the stimulatederythropoiesis enhancement in the rHuEPO-Fc-treated macaques wasobserved (the mean RBC numbers increased from 4.74×10⁹/ml to 6.67×10⁹/mland the mean Hb levels from 12.2 g/dl to 13.7 g/dl), rHuEPO-Fc failed toinduce detectable specific antibodies against the fusion protein. Theseresults indicate that rHuEPO-Fc fusion protein does not causeimmunogenicity in primates.

9. Acute Toxicity Studies of rHuEPO-Fc in Normal Mice

To assess the safety of rHuEPO-Fc fusion protein, acute toxic studieswere conducted in animals.

Two groups of BALB/c mice (n=20, equal numbers of male and female, 5-6weeks old, the average weight of female is 15.8±0.4 g, male is 15.9±0.6g, purchased from Chinese Academy of Medicine, China) were injectedintravenously once with excessive amount of purified rHuEPO-Fc(male=13.3 mg/kg, female=13.2 mg/kg) or equal volume of the carriersolution via their tail veins respectively. In addition to observing theinstant reaction following injection, general behavior and status,activities, eating and defecation patterns and changes were monitoredand recorded daily for 14 days. All mice were also weighed at day 7 andday 14. At day 15 post-injection, the anatomic examination of the mainorgans of the mice were conducted. Pathologic examination would beconducted if any unusual changes or suspicious changes of the organswere observed.

All mice in the 2 groups had no obvious instant reaction followinginjection. Within the period of 14 days, no obvious changes of behavior,activities, eating and defecation patterns were observed. Moreover, theweight of the mice in both groups increased steadily during the testingperiod, and no apparent differences were found between the 2 groups onday 7 or day 14 post injection. No abnormal or pathologic changes weredetected in the tissues of brain, lung, heart, liver and kidney. Theseresults indicate that administration of excessive amount of rHuEPO-Fc,far more than required for exhibiting the normal erythropoiesisfunction, is safe and had no apparent toxic effects.

As will be apparent to those skilled in the art in the light of theforegoing disclosure, many alterations and modifications are possible inthe practice of this invention without departing from the spirit orscope thereof.

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1. An isolated polynucleotide having a nucleic acid sequence thatencodes a fusion protein having at least 90% amino acid sequenceidentity to SEQ ID NO: 2, said polynucleotide encoding a non-cysteineamino acid at residue 172 of said amino acid sequence, wherein saidprotein has a prolonged half-life in vivo in comparison to native humanerythropoietin.
 2. The polynucleotide as defined in claim 1, whereinsaid nucleic acid sequence encodes a protein having at least 98% aminoacid sequence identity to SEQ ID NO:
 2. 3. An isolated polynucleotidecomprising a nucleic acid sequence having at least 90% sequence identityto SEQ ID NO: 1 and encoding a fusion protein having an in vivoerthryopoietic potency at least equal to native human erythropoietin andhaving a prolonged half-life in vivo in comparison to native humanerythropoietin.
 4. The polynucleotide as defined in claim 3 having atleast 95% sequence identity to SEQ ID NO:1.
 5. A polynucleotide asdefined in claim 4 having at least 98% sequence identity to SEQ ID NO:1.6. An isolated recombinant DNA molecule comprising the nucleic acidsequence present in SEQ ID NO: 1 or a sequence having 98% sequenceidentity thereto.
 7. A cell line transfected with a recombinant DNAmolecule as defined in claim
 6. 8. The cell line as defined in claim 7,wherein said cell line is a CHO cell line.
 9. A method of producing aprotein as defined in claim 1, comprising culturing a cell linetransfected with the DNA molecule as defined in claim 6 and purifyingthe protein encoded thereby.
 10. An isolated recombinant DNA moleculecomprising the nucleic acid sequence present in SEQ ID NO:
 1. 11. Anisolated nucleic acid molecule having a sequence that encodes apolypeptide with at least 98% amino acid sequence identity to SEQ ID NO:2, wherein said polypeptide has a prolonged half-life in vivo incomparison to native human erythropoietin, wherein said polypeptidecomprises a fragment of a human immunoglobulin molecule whose N-terminalis directly linked to a C-terminal of an erythropoietin molecule, andwherein a hinge region of said immunoglobulin molecule has a mutationwhereby a cysteine residue nearest said N-terminal is replaced with anon-cysteine residue.